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2. Monoclonal antibody preparations contain a homogeneous population of identical antibody molecules with identical binding sites. They have a well-de ned interactions between this site and a single antigen epitope. This results in a linear Scarchard plot. Polyclonal preparations consist of a mixture of antibody molecules, some reacting with antigen. Those that bind antigen possess a number of different paratopes, so that a variety of antigen epitopes bind, each with a different af nity. This results in a curved Scatchard plot. 3. (a) No precipitin forms. Anti-B, -C, or -D must also be present to obtain precipitin. (b) Precipitin is present since antigen is multivalent. (c) No precipitin forms because only the primary Ag:Ab reaction can occur. (d) Precipitin forms [same argument as for (b)]. 4. Yes, the bacterium was present, since an agglutination zone was observed. A prozone is only observed if the antigen is present in excess, at the low dilution (concentrated) end of the dilution series; since no prozone was observed, the bacterium was initially present at too dilute a concentration for the observation of a prozone. 5. The precipitin line would curve toward the antigen well. CHAPTER 6 1. (a) Estrogen, (b) human a-fetoprotein, (c) mouse antibody, and (d) acetylcholine. 2. (a) Antibody, (b) antibody, and (c) labeled ligand and ligand/unknown. 3. Fluorophore Chemiluminescent Enzyme Cofactor Lysing agent Secondary label Prosthetic group Electroactive Spin label Substrate Continuous Single event (e.g., 1 photon/reaction) Catalytic ampli cation Continuous (like substrate) Catalytic ampli cation (trapped E) Catalytic ampli cation (binds E) Catalytic ampli cation (creates active E) Single event (e.g., 1 electron/ferrocene) Continuous Continuous (e.g. A of product)
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4. (a) Ab is present at limited concentration, while H, H and Ab* are all H, present in excess. (b) Only one other species will be present, Ab H. (c) Of these potential interferents, E is expected to cross-react to the greatest extent, since it is the most identical to C, especially in the region most likely to comprise the epitope. The least likely to interfere is D, since it is different in the Ab-binding region.
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(d) Plot is sigmoid, with signals high at low [testosterone] and decreasing as concentration increases. 5. (a) Plot is sigmoid, with higher signals at low [free biotin] values, decreasing as [biotin] increases. (b) [Substrate] > 1.0 mM should be used (10 Km ). (c) Ideally, the plot of light intensity, or photons detected, as a function of time will be at, that is, photons are emitted and detected at a constant rate. In contrast, absorbing products accumulate in an enzymatic reaction, so that absorbance increases with time. 6. This assay is noncompetitive, so that linear emission concentration (rather than sigmoid emission log[Ag]) plots are observed. Linear log log plots will also be observed, and as shown in the Figure 6.6, F1 emission decreases with increasing concentration while F2 emission increases with increasing Ag. CHAPTER 7 1. (a) i 26 mA; do not forget to convert cubic centimeter (cm3) units to liters (L). (b) Same as (a) current does not change. 2. The assumption is invalid when very small sample volumes are used. Then analyte is consumed, and the concentration of analyte in the reaction layer decreases with time. 3. We want NADPH to be produced very close to the surface of the optical ber, so that the evanescent wave will detect it. Option (a) is not the best, since adsorption would lead to a loss of activity, and the monolayer would have a low total activity. Option (c) also produces a monolayer, and may not fully convert glucose (i.e., [S]o does not equal 0). Option (b) looks the best, since complete conversion of glucose would occur in the outer layers, and the production of NADPH occur in the outer layers, and the production of NADPH occurs at the surface of the ber. 4. Thermal sensors rely on heat retention, to measure heat generated by the enzymatic reaction. This will result in denaturation over the longer term. Amperometric sensors are not insulated, so that heat dissipation is more ef cient. CHAPTER 8 1. False. Biological and analytical gures of merit are not always the same. 2. The main criterion is the availability of knowledge to guide a rational design process. Secondary criteria would include the lack of an available method to screen a large library, and the nature of the desired improvement.
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